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Abstract

Background

Widespread sampling of vertebrates, which comprise the majority of published animal
mitochondrial genomes, has led to the view that mitochondrial gene rearrangements
are relatively rare, and that gene orders are typically stable across major taxonomic
groups. In contrast, more limited sampling within the Phylum Mollusca has revealed
an unusually high number of gene order arrangements. Here we provide evidence that
the lability of the molluscan mitochondrial genome extends to the family level by
describing extensive gene order changes that have occurred within the Vermetidae,
a family of sessile marine gastropods that radiated from a basal caenogastropod stock
during the Cenozoic Era.

Results

Major mitochondrial gene rearrangements have occurred within this family at a scale
unexpected for such an evolutionarily young group and unprecedented for any caenogastropod
examined to date. We determined the complete mitochondrial genomes of four species
(Dendropoma maximum, D. gregarium, Eualetes tulipa, and Thylacodes squamigerus) and the partial mitochondrial genomes of two others (Vermetus erectus and Thylaeodus sp.). Each of the six vermetid gastropods assayed possessed a unique gene order. In
addition to the typical mitochondrial genome complement of 37 genes, additional tRNA
genes were evident in D. gregarium (trnK) and Thylacodes squamigerus (trnV, trnLUUR). Three pseudogenes and additional tRNAs found within the genome of Thylacodes squamigerus provide evidence of a past duplication event in this taxon. Likewise, high sequence
similarities between isoaccepting leucine tRNAs in Thylacodes, Eualetes, and Thylaeodus suggest that tRNA remolding has been rife within this family. While vermetids exhibit
gene arrangements diagnostic of this family, they also share arrangements with littorinimorph
caenogastropods, with which they have been linked based on sperm morphology and primary
sequence-based phylogenies.

Conclusions

We have uncovered major changes in gene order within a family of caenogastropod molluscs
that are indicative of a highly dynamic mitochondrial genome. Studies of mitochondrial
genomes at such low taxonomic levels should help to illuminate the dynamics of gene
order change, since the telltale vestiges of gene duplication, translocation, and
remolding have not yet been erased entirely. Likewise, gene order characters may improve
phylogenetic hypotheses at finer taxonomic levels than once anticipated and aid in
investigating the conditions under which sequence-based phylogenies lack resolution
or prove misleading.

Background

Animal mitochondrial (mt) genomes typically consist of a circular molecule of DNA
encoding 37 genes (2 rRNA genes, 13 protein-encoding genes, and 22 tRNA genes), the
arrangement of which is often highly conserved within major taxonomic groups [1]. Consequently, when gene rearrangements occur, they may provide compelling phylogenetic
markers that can corroborate or contradict hypotheses based on primary sequence data
and provide resolution for deeper nodes that are often weakly supported in sequence-based
phylogenies [2-6]. With recent technological and methodological advances (e.g., rolling circle amplification:
[7,8]; next generation sequencing technologies: [9]), and associated decreasing costs of DNA sequencing, the amplification and sequencing
of whole mt genomes has become routine. As a result, there has been a marked increase
in the sequencing of whole animal mt genomes over the past decade as well as the development
of computational methods to extract phylogenetic information from these genomes through
inferences of past gene dynamics [10-12]. To date, 1868 complete metazoan mt genomes are available in the NCBI Genomes database
http://www.ncbi.nlm.nih.gov/guide/genomes/webcite; January 8, 2010), the majority belonging to arthropods (293) and vertebrates (1292).

Compared to other major metazoan phyla, molluscan mitochondrial genomes are poorly
represented at NCBI [13], with only 78 complete mt genomes available as of January, 2010. Despite this, molluscan
mt genomes are beginning to challenge the traditional view that mitochondrial gene
orders are stable over long periods of evolutionary time [13-16], a view based largely on the heavily sampled and highly conserved mt genomes of vertebrates.
Instead, mollusc mt genomes demonstrate substantial heterogeneity in length and "architecture"
[16], reflecting differences in gene complement resulting from gene loss or duplication,
as well as changes in the position and strand specificity of tRNA genes, protein-encoding
genes, and rRNA genes. Changes in gene arrangement within the Mollusca have been so
dramatic that representatives of four classes of molluscs (Gastropoda; Bivalvia; Cephalopoda;
Scaphopoda) share remarkably few mitochondrial gene boundaries, with gene orders varying
extensively even across major lineages of bivalves as well as gastropods [14]. Changes in gene arrangement have also been observed within bivalve and gastropod genera, based on changes in position of: 1) tRNAs and an rRNA
gene in the oyster, Crassostrea [17], and 2) protein encoding and tRNA genes in the vermetid marine gastropod genus, Dendropoma [18]. Differences in gene order are also evident between paternally versus maternally
inherited mitochondrial genomes of bivalves exhibiting doubly uniparental inheritance
[19], including the unionid freshwater bivalve, Inversidens japanensis [14], and the marine venerid clam, Venerupis (Ruditapes) philippinarum (NCBI, unpublished). Similar intrageneric gene translocations have now been described
in 19 of 144 genera in which two or more complete mt genomes have been sequenced [16], including representatives of the Porifera, Platyhelminthes, Nematoda, Mollusca,
Arthropoda and Chordata. Thus, growing evidence suggests that mt genomes of many metazoan
phyla may be considerably more plastic than originally believed, with the conserved
genome architecture of vertebrates reflecting a derived stabilization of the mt genome
and not an ancestral feature [16].

The discovery of mt gene order changes at lower taxonomic levels, as found within
the Mollusca, is exciting for several reasons. First, gene dynamics involving translocations
and inversions of genes offer the promise of new and robust characters that can be
used to support phylogenetic hypotheses at the level of families, genera, and species
[18]. Given the comparatively low rate of rearrangement and the astronomical number of
possible gene arrangements, convergence is likely to be rare compared to four-state
nucleotide sequence data [20]. Second, it is becoming increasingly apparent that the application of mitochondrial
sequences and gene order data to questions of evolutionary history and phylogenetic
relatedness requires a better understanding of the evolutionary dynamics of mt genomes
[21]. Basic mechanisms of gene rearrangement associated with slipped-strand mispairing
[22], errors in replication origins or end points [23], and intramolecular recombination [24], remain poorly understood. Likewise, tRNA remolding and tRNA recruitment events [25-28], gene rearrangement "hotspots" [29,30], the non-random loss of duplicated genes [31] and gene order homoplasy [32-34], which can act to confound phylogenetic inferences based on mtDNA sequences and gene
orders, need to be explored more fully. Comparison of gene arrangements at low taxonomic
levels can help to elucidate the process of gene rearrangement. For instance, the
signature of specific processes such as tRNA remolding or recruitment can be most
easily recognized when such events have occurred recently, since remolded or recruited
tRNAs can be identified through high similarity scores and phylogenetic analyses [27,28]. Likewise, those taxonomic groupings with unusually labile genomes offer the opportunity
to investigate the mechanics of gene rearrangement: telltale vestiges of gene duplication
and translocation, typically erased or overwritten with time, may still be present
within these genomes [35] and such intermediate stages can be critical to reconstructing the processes through
which such gene rearrangements have occurred. Comparisons of mt genomes at low taxonomic
levels, even within families and genera, can thus be extremely helpful in interpreting
the evolutionary dynamics of these genomes and exploiting the phylogenetic signal
retained within these DNA molecules [16].

Here we present further evidence of highly dynamic molluscan mt genomes by revealing
extensive gene order changes within members of one caenogastropod family: the Vermetidae.
Vermetids are a group of sessile, irregularly coiled, suspension-feeding gastropods
found in warm temperate to tropical oceans around the world that radiated from a basal
caenogastropod stock in the early Cenozoic Era. They are currently classified as members
of the Hypsogastropoda [36,37], a large and diverse group with a fossil record extending back to the Permo-Triassic
boundary that includes all extant caenogastropods, except for the Architaenioglossa,
Cerithioidea and Campaniloidea. While relationships within the Hypsogastropoda are
not well resolved, vermetids are typically positioned within the infraorder Littorinimorpha.
More specifically, molecular analyses suggest that vermetids are members of a largely
asiphonate clade of gastropods including the Littorinidae, Eatoniellidae, Rissoidae,
Anabathridae, Hipponicidae, Pterotracheidae, Epitoniidae, Cerithiopsidae, Eulimidae,
and Naticidae [37]. This association is also supported by morphological similarities in euspermatozoa
shared by many members of this clade [38].

Gene order rearrangements have been recognized previously in this family [18] based on small (<3.5 kb) portions of the mt genome sequenced from several species
within the genus Dendropoma. In this paper, we expand upon these earlier results by providing complete mt genomes
for two Dendropoma species as well as for representatives of two other vermetid genera, Thylacodes and Eualetes. We also reveal additional gene rearrangements within this family through the partial
genomes of the vermetid genera Thylaeodus and Vermetus. The extent of gene rearrangement within the family offers great potential for improving
our phylogenetic hypothesis for the enigmatic Vermetidae as well as for understanding
more fully the mechanics of gene order change within metazoan mt genomes.

Methods

Amplification and sequencing

Selection of taxa was based on: 1) the discovery of novel gene orders in these species
following PCR amplifications spanning gene boundaries [18] and 2) those genomes that successfully amplified using long and accurate PCR (LAPCR).
The collecting locality, tissue source, and Field Museum of Natural History (FMNH)
voucher information for each specimen, are presented in Table 1, along with GenBank accession numbers, primer sequences, and lengths of amplification
products. Other gene arrangements have been identified in additional vermetids based
on partial genome sequences (<3 kb), but these results are not presented here (Rawlings
et al., in prep).

Table 1. Sampling localities, tissue sources, and PCR primer sequences for the six vermetid
species examined in this study

DNA was extracted from ethanol-preserved tissues using a phenol chloroform extraction
protocol as described in [39]. Initially, an 1800 bp region of the mitochondrial genome spanning the rrnS to nad1 region was amplified as part of a phylogenetic analysis of Vermetidae [18]. This sequence was subsequently used to design outwardly facing primers for LAPCR
(Table 1: rrnL-F; rrnL-R). Because attempts to amplify the genome in one piece were not successful,
we amplified a 650 bp fragment of cox1 using modifications of Folmer's widely used cox1 primers [40]. This cox1 sequence was then used as a template for designing a second pair of primers (cox1-F;
cox1-R). Successful amplifications were associated with the primer combinations: rrnL-F/cox1-R
("A" fragment) and rrnL-R/cox1-F ("B" fragment). LAPCR reactions were undertaken using
GeneAmp XL PCR kits (Applied Biosystems; N8080193). Typically, 25 μL reactions were
set up in two parts separated by a wax bead following the manufacturer's recommendations,
using a [Mg(OAc)2] of 1.2 mM and an annealing temperature specific to the primer combination. Typical
conditions consisted of a 94°C denaturation period lasting 60 s, followed by: 16 cycles
at 94°C for 25 s, 60°C for 60 s, and 68°C for 10 min; 18 cycles at 94°C for 25 s,
60°C for 60 s, and 68°C for 12 min; and a final extension period at 72°C for 10 min.
Amplifications were run on a Stratagene Gradient Robocycler. PCR products were cleaned
by separating high molecular weight products from primers and sequencing reagents
using Millipore Ultrafree filter columns [7]. Samples were added to 200 μL of sterile water and then spun in a picofuge for 15
min or until the filter membranes were dry. PCR products were eluted from the membrane
in 20 μL of sterile distilled water, and 5 μL of this product was run out on a 0.8%
agarose gel to confirm the presence of a band of the appropriate size. Typically,
products from several replicate PCR reactions were pooled prior to quantitation. Once
3 ng of PCR product had been obtained, samples were dried down in a vacuum centrifuge
and sent to the Joint Genome Institute, Walnut Creek, CA, where they were sequenced
using standard shotgun sequencing protocols [7,8].

Genome annotation and analysis

Genome annotation

The approximate locations of the rRNA and protein-encoding genes were determined by
aligning each unannotated genome with genes from other caenogastropod mt genomes.
The precise boundaries of rRNA genes could not be determined due to the lack of sequence
similarity at the 5' and 3' ends; therefore, the location of each rRNA gene was assumed
to extend from the boundary of the upstream flanking gene to the boundary of the downstream
flanking gene, as in [41]. A standard initiation codon was located at the beginning of each protein-encoding
gene (either ATG, ATA, or GTG), and the derived amino acid sequence was aligned with
homologous protein sequences to ensure that this was a suitable initiation codon (based
on length). When possible, the first proper stop codon (TAG or TAA) downstream of
the initiation codon was chosen to terminate translation; however, to reduce overlap
with downstream genes, abbreviated stop codons (T or TA) were selected for some genes.
In these instances, polyadenylation of the mRNA was assumed to restore a full TAA
stop codon [7]. Regardless, some genes appeared to overlap based on the conservation of their open
reading frame sequences and the lack of a potential abbreviated stop codon. Gene locations
and secondary structures of tRNAs were identified using tRNAscanSE [42] and ARWEN [43]. On the rare occasion that these programs did not find all of the expected tRNAs,
the remaining tRNAs were found by eye and folded manually. We produced tRNA drawings
manually using Canvas (ACD Systems). Using cox1 as the conventional starting point for the four genomes, linear maps of the circular
genomes were created to facilitate comparison of gene orders amongst vermetids, across
available caenogastropods, and between caenogastropods and other select molluscs,
including the abalone, Haliotis rubra (Class Gastropoda,Superorder Vetigastropoda), the octopus, Octopus vulgaris (Class Cephalopoda), and the chiton, Katharina tunicata (Class Polyplacophora).

Nucleotide composition

The nucleotide composition of each complete and partial genome was described by calculating
the overall base composition, %AT content, AT-skew, and GC-skew for the strand encoding
cox1, hereafter referred to as the "+" strand. Base composition and %AT content were determined
using MacVector (MacVector, Inc.), and strand skews (AT skew = (A-T)/(A+T); GC skew
= (G-C)/G+C)) were calculated using the formulae of [44]. For complete genomes, the %AT content, AT-skew, and GC-skew were also calculated
for rRNA genes, protein-encoding genes (for all bases, third codon positions, and
third positions of four-fold degenerate (4FD) codons, as in [44]), tRNA genes (separately for those coded for on the "+" and "-" strand), and intergenic
(unassigned) regions. Values were compared across categories within each genome, and
within categories across genomes. In addition, we explored the nucleotide composition
at third positions of 4FD codons in relation to the position of each protein-encoding
gene within the genome [45]. Gene positions were determined by calculating the distance (number of nucleotides)
from the midpoint of each gene to a reference point chosen here as the start of nad1.

A base composition plot (%A+C and %G+T along the length of the genome) was created
for each complete genome using a sliding window of 100 nucleotides. Typically, the
leading strand is G+T rich associated with its protracted single-stranded state during
replication and transcription; deviations from this pattern can signify switches in
the assignments of these strands [41]. Plots were aligned with linear representations of the matching genome to signify
base composition trends for protein and tRNA genes encoded on the "+" strand, tRNA
genes encoded on the "-" strand, and rRNA genes.

Genetic code

To ensure that all codons were being utilized, codon usage frequencies were analyzed
using MacVector (MacVector, Inc.); unused codons can potentially signal a change in
the genetic code [46]. Codons whose amino acid identity has changed in the mt genetic code of other metazoans
(e.g. flatworms, echinoderms, and hemichordates; [47]) were investigated using a procedure similar to [48]: the derived amino acids of five codons (TGA, ATA, AGA, AGG, AAA) were examined in
the alignment of three highly conserved proteins (cox1, cox2, and cox3) from the mt genomes of four vermetids, D. gregarium, D. maximum, E. tulipa and T. squamigerus, and several other caenogastropods. Within the vermetids, if the derived amino acid
from any of these five codons occurred in a conserved position (present at that location
in >50% of caenogastropod sequences examined) this was scored as a positive result.
Amino acids that occurred in a non-conserved position (<50%) were scored as negative.
The proportion of appearances in conserved vs. non-conserved locations was calculated for each codon across the vermetids examined.
High percentages in conserved locations likely indicate the retention of amino acid
identity by the specific codon [48]; low percentages can be suggestive of a change in amino acid identity, although other
explanations are also possible.

Unassigned regions

As metazoan mt genomes are typically compact with minimal non-coding DNA [49], unassigned stretches of nucleotides often contain control elements for transcription
or replication or remnants of duplicated protein, rRNA or tRNA genes. We examined
putative non-coding regions (> 20 bp) for gene remnants, repeat sequences, inverted
repeats, palindromes, and secondary structure features, all of which can be associated
with signaling elements of the mt genome. Protein, rRNA and tRNA remnants were identified
by aligning unassigned regions with annotated genes from caenogastropod mitochondrial
genomes. MEME [50] and M-Fold [51] were used to identify potential sequence motifs and structural features, respectively.

tRNA remolding/recruitment

To search for close matches between tRNA genes indicative of gene remolding/recruitment,
each tRNA gene sequence was aligned to tRNA genes from other available caenogastropod
genomes (including new vermetid tRNA sequences). Similarity scores were based on initial
tRNA alignments undertaken in MacVector that were subsequently adjusted by eye according
to secondary structure features (stems vs. loops). As in [28], the third base of the anticodon triplet was excluded in the calculation of % similarity
between two tRNA genes, but gaps were counted as mismatches.

Results and Discussion

Caenogastropod mitochondrial genomes

To date, comparisons across published caenogastropod mt genomes have suggested a model
of gene order conservation unusual for the Gastropoda [15,48,52,53]. Among the 16 complete caenogastropod genomes available at NCBI as of January 8,
2010, gene order rearrangements within this clade appear minor, involving only changes
in position of trnLUUR, trnLCUN, trnV and trnS2, and an inversion of trnT ([53]). Likewise, only two rearrangements, one inversion and one transposition, separate
the reconstructed ancestral gastropod gene order from that of most caenogastropods
[15,53]. While this conservation in gene order within the Caenogastropoda may be real, it
could also reflect a strong sampling bias for members of the Neogastropoda (12 complete
genomes) - a largely Cenozoic radiation of predatory snails - with only four complete
genomes from two genera (Cymatium and Oncomelania), examined from other caenogastropod groups. In contrast, wider sampling of mt gene
orders within the Heterobranchia, a sister clade to the Caenogastropoda, has demonstrated
highly dynamic mt genomes [15]. Of 13 genomes sampled across a disparate array of taxa, including 5 opisthobranchs
(including an unpublished Elysia genome), 7 pulmonates (including a second Biomphalaria genome not included in [15]), and 1 basal heterobranch [15], numerous changes in gene order have been observed, with few mt gene boundaries shared
between heterobranchs and the hypothetical ancestral gastropod mt genome inferred
by [15].

Here, based on a detailed sampling of mt genomes within one family of caenogastropods
outside the Neogastropoda, we provide a new and very different picture of gene order
dynamics within the Caenogastropoda. Our results increase the number of caenogastropod
mt genomes sequenced to 20, substantially increase the sampling of mt genomes outside
the Neogastropoda, and present the first direct evidence of major gene order rearrangements
within the Littorinimorpha based on complete genome sequences. Full mt genomes were
successfully sequenced for the vermetids, Dendropoma gregarium, D. maximum, Eualetes tulipa, and Thylacodes squamigerus (Figure 1; Tables 2, 3, 4 and 5). Amplifications of the "B" fragment (cox1-F/rrnL-R) were not successful for Thylaeodus sp. and V. erectus and consequently only partial mt genomes are described for these two taxa (Figure
1; Tables 6 and 7). Nevertheless, extensive gene rearrangements were evident within these species compared
to other vermetids and caenogastropods. Characterization of the vermetid mt genomes
presented below is based only on the four complete genomes, except where noted otherwise.

Figure 1.Gene maps of four complete and two partial vermetid mt genomes. Blue arrows represent protein-encoding genes; red arrows represent rRNA genes; and
circles represent tRNA genes. Arrows indicate the direction of transcription (clockwise
= "+" strand). Genes are labeled according to their standard abbreviations. For tRNA
genes, green circles represent those genes located on the "+" strand and white circles
those located on the "-" strand. Regions of unassigned nucleotides (presumed non-coding
sequences) are not identified on these maps. As is the standard convention for metazoan
mt genomes, cox1 has been designated the start point for the "+" strand. The asterisks (*) in D. maximum and T. squamigerus denote stretches of sequences (presumed pseudogenes) with high similarity to portions
of complete genes. For exact locations of each gene, as well as unassigned regions,
see Tables 2-7.

Genome organization

The mitochondrial genomes of D. gregarium, D. maximum, E. tulipa, and T. squamigerus varied in size from 15078 bp to 15641 bp, similar in length to other caenogastropods
(range: 15182 - 16648 bp, n = 16) but considerably larger than most heterobranchs
(range: 13670 - 14745 bp, n = 13). Each genome contained the 37 genes typical of most
animal mitochondrial genomes (Figure 1; Tables 2, 3, 4 and 5), with all 13 protein-encoding genes and 2 rRNA genes located on the "+" strand along
with either 14 or 15 tRNA genes depending on the species. In each of the four genomes,
the tRNA genes, trnY, trnM, trnC, trnW, trnQ, trnG, and trnE, were located on the "-" strand forming a cassette of 7 adjacent tRNAs. The only
difference across genomes in the strand specific coding of a gene was for trnT, which was located on the "+" strand of E. tulipa, but on the "-" strand of D. gregarium, D. maximum, and T. squamigerus. The tendency for protein and rRNA genes to be coded for on the same strand has been
found in all caenogastropod taxa examined so far, but this is atypical for molluscs
(excluding bivalves) and other metazoan mt genomes described to date (137 of 1428
genomes as of Dec 17, 2008; [54]). The predominance of this single-strand-dependence for protein and rRNA genes among
sponges and cnidarians has led to the proposition that this is the plesiomorphic metazoan
condition [54].

The tRNA genes identified within each genome and their secondary structures are presented
as additional files (see Additional file 1, Figure S1; Additional file 2, Figure S2; Additional file 3, Figure S3; Additional file 4, Figure S4; Additional file 5, Figure S5; Additional file 6, Figure S6). Extra tRNA genes were found in two vermetid genomes. D. gregarium had a second trnK located between genes trnI and nad3 (see Figure 1). This second trnK can be folded in to a typical tRNA cloverleaf structure (see Additional file 2, Figure S2), and thus may be functional. The genome of T. squamigerus also contained additional trnV and trnLUUR genes (see Figure 1). These genes were associated with two large stretches of unassigned sequence positioned
between the two copies of trnV (see below).

Additional file 1.Figure S1. Inferred tRNA secondary structures based on the nucleotide sequences of 22 mitochondrial
tRNA genes identified from the complete mt genome of Dendropoma maximum. tRNA genes are labeled according to their amino acid specificity and are arranged
alphabetically.

Additional file 6.Figure S6. Inferred tRNA secondary structures based on the nucleotide sequences of five mitochondrial
tRNA genes identified from the partial mt genome of Vermetus erectus. tRNA genes are labeled according to their amino acid specificity and are arranged
alphabetically.

Overlapping adjacent genes were common in vermetid mt genomes. Two pairs of protein-encoding
genes overlapped in all four species (atp8 and atp6; nad4L and nad4); overlap of nad4L and nad4 was also evident in the partial genome of Thylaeodus. These gene pairs commonly overlap in animal mt genomes. This phenomenon was more
variable between tRNA genes (see Tables 2, 3, 4, 5, 6 and 7 for specific examples). In addition, in three instances tRNA genes overlapped with
protein-encoding genes (trnH and nad4 in E. tulipa; trnSAGN with cox3 and trnR with nad2 in T. squamigerus). Similar comparisons could not be made for the two rRNA genes since their boundaries
were only imprecisely defined by boundaries with neighbouring genes.

Gene initiation and termination

Nearly all protein-encoding genes (58/61) were initiated by the canonical ATG start
codon, although ATA (twice) and GTG (once) also acted as start codons (Tables 2, 3, 4, 5, 6 and 7). These start codons are not unusual in molluscan mt genomes, but their usage, as
found elsewhere, appears to be much less frequent than ATG [7] (but see [15]). TAG was the most common termination codon (28/59), but TAA (15/59) and abbreviated
stop codons (a single T or TA terminating the open reading frame; 16/59) were used
slightly more frequently when considered together. The sequences for the cox1 genes of Thylaeodus and Vermetus were incomplete so the termination codons for these genes are currently unknown.

Unassigned regions

The number of nucleotides unassignable to any gene ranged from 289 (1.9% of the genome)
in Eualetes tulipa to 625 (4.0% of the genome) in Thylacodes squamigerus (Table 8). No unassigned stretch of sequence was greater than 270 bp in length, although intergenic
regions ranging in size from 10 - 99 bp were common (Table 8). There was little consistency in size and position of unassigned regions across
genomes, with the position of the largest region found in different areas for each
species. Short (≤ 15 bp) AT-rich (>80%) intergenic regions were evident within the
genomes of Dendropoma maximum, D. gregarium, and T. squamigerus, but not in E. tulipa (Tables 2, 3, 4 and 5), and again, the position of these sequences varied across taxa. Stretches of unassigned
sequence including inverted repetitive elements are known to reside between trnF and cox3 in the mt genomes of several caenogastropods, likely representing the control region
for replication and transcription in these genomes [52,53]. In our comparisons across the four complete vermetid genomes, we were unable to
identify any similar regions associated with repetitive sequence motifs or palindromes.
As is common in mt genomes, however, secondary structure elements were found by MFOLD
within many of these intergenic regions, suggestive of a role in post-transcriptional
modification of the polycistronic transcript.

Table 8. A comparison of unassigned regions (UR) within the complete mt genomes of four vermetid
gastropods

Our analyses did uncover several interesting features within large intergenic regions
of Dendropoma maximum and Thylacodes squamigerus (Figure 1). In D. maximum, we identified a 121 bp stretch within an unassigned region of 167 bp between trnR and trnY that was a perfect match to the reverse complement of a portion of rrnS. Likewise, in Thylacodes squamigerus, in an unassigned region between trnV1 and trnLCUN, we found a 140 bp stretch of sequence that was identical to a corresponding portion
of rrnL. In addition, between trnLCUN and trnV2, we discovered a 120 bp remnant of nad1 (82% identical, with a 91 bp stretch differing only in 6 bases) and a 114 bp remnant
of rrnS (92% identical). These pseudogene fragments in Thylacodes, as well as the extra tRNAs (trnV and trnLUUR) present in this region, are likely the result of a duplication event spanning rrnS-trnV-rrnL-trnLUUR-trnLCUN-nad1, with subsequent overwriting of some gene duplicates (Rawlings et al., in prep).

Nucleotide composition and skews

All four vermetid genomes were AT-rich, with these two nucleotides accounting for
59 - 62% of the genome (Figure 2; Additional file 7, Table S1); this trend was consistent across regions of each genome associated with
protein-encoding genes, rRNA genes, tRNAs, and non-coding regions. Interestingly,
however, the AT-biases of vermetid genomes were noticeably lower than those reported
for other complete caenogastropod genomes (range: 65.2-70.1%, n = 14; Additional file
7, Table S1). Likewise, AT content was only moderately higher at third-codon positions
and 4FD sites of protein-encoding genes, a result unusual in comparison with many
other protostomes where %AT can often exceed >80% at 4FD sites [44]. Skew analyses revealed that the "+" strand was strongly biased against A (AT-skew
ranging from -0.148 to -0.238) and towards G (GC-skew ranging from +0.065 to +0.251).
This pattern was similar to other caenogastropods (Additional file 7, Table S1), heterobranchs [15], and the chiton, Katharina tunicata [55], but opposite to that found on the "+" strand of the abalone Haliotis [56] and the cephalopods, Nautilus and Octopus, suggestive of switches in the assignments of leading and lagging strands within
the Mollusca [41].

Skew patterns were different between regions of the genome depending on their function.
For AT-skews, the most strongly biased areas were found associated with protein-encoding
genes, but these were not necessarily most pronounced at third positions or at 4FD
sites (except for D. maximum; Figure 2). The two rRNA genes showed a marked difference from protein-encoding genes, with
a slight positive AT-skew, on average. Differences in skew patterns between rRNA and
protein-encoding genes have been noted elsewhere [41,57], and may reflect base-pairing constraints associated with the secondary structures
of these rRNAs [41]. Unassigned regions, assumed to be non-coding, were expected to experience similar
selective pressures to those of 4FD third codon positions; this correspondence was
not strong, however, with unassigned regions showing variation in AT-bias across taxa
from slight positive AT-skews to moderate negative skews. tRNA genes regardless of
whether they were coded for on the "+" or "-" strand exhibited no consistent skew
patterns, with an average close to zero.

GC-skews were moderately positive for most regions of the genome, including protein-encoding
genes, rRNA genes, unassigned regions, and tRNAs encoded on the "+" strand. Skews
were particularly strong, however, for the third codon positions and 4FD third codon
positions in Dendropoma maximum and Eualetes tulipa. On average, neutral to negative GC-skews were associated with regions of the "+"
strand associated with tRNA genes encoded on the "-" strand.

As predicted by skew patterns described above, G+T composition varied across the length
of the "+" strand in all four complete genomes (Figure 3). Areas of weak or no G+T bias were typically associated with rRNAs and regions with
tRNAs encoded on the "-" strand. Dendropoma maximum exhibited a particularly strong bias for G+T (>65%) in the region from 10,000 to 14,000
bp.

Figure 3.Plots of A+C and G+T composition along the "+" strand of the mt genomes of D. maximum, D. gregarium, E. tulipa, and T. squamigerus using a sliding window of 100 nucleotides. Each plot is positioned above a linear representation of its corresponding genome
highlighting regions corresponding to the presence of: 1) protein encoding genes and
tRNA genes encoded on the "+" strand, 2) rRNA genes encoded on the "+" strand, and
3) tRNA genes encoded on the "-" strand. See [41] for a comparison with Nautilus and Katharina. As is the standard convention for metazoan mt genomes, cox1 has been designated the start point for the "+" strand.

Codon frequency

No evidence was found of changes to the standard genetic code employed by other molluscs
(Additional file 8, Table S2). Each codon was used multiple times in the protein-encoding genes of each
species (Additional file 9, Table S3), however, codon usage frequencies were not equal. Codon usage strongly
reflected skew patterns at the third position for synonymous codons (both two-fold
and four-fold degenerate codons; see Additional file 9, Table S3): the most abundant base at the third codon position across all four complete
genomes was T (36.7-43.1%) and the least abundant base was C (9.4-17.9%). The relative
ranking of G (18.5-26.5%) and of A (21.0-29.8%), however, was less consistent across
genomes.

Putative origins of replication

Locating the regions of the mt genomes associated with the replication origins (ORs,
also known as control regions or A+T rich regions) for both "+" and "-" strands can
be challenging based on a knowledge of the nucleotide sequence of mt genomes alone.
Recognition elements can be in the form of conserved sequence blocks, regions rich
in A+T, stable stem-loop structures containing T-rich loops, or repetitive elements/palindromes
[58]. ORs can also be identified by association with rearrangement "hotspots" [23]. Such features are not definitive evidence, however, and can differ in utility across
taxonomic groups. For instance, only 35% of tentatively identified molluscan control
regions have been found to have palindromes and the average %AT content of these regions
is in the range of 65-70%, close to the average genomic %AT content for molluscs (based
on 9 molluscan genomes analyzed) [59]. Consequently, looking for palindromes or A+T rich regions may not be helpful in
identifying the OR of molluscs. In contrast, 85% of insect control regions have palindromes
and CR regions are associated with >80% AT [59].

Because none of the recognition elements described above were useful in locating putative
ORs within the complete genomes of the four vermetids examined here, we attempted
to locate ORs by examining the relationship between nucleotide composition and the
position of protein-encoding genes within these genomes. Reyes et al. [45] determined that the AT and GC-skews at 4FD sites of protein-encoding genes of 25
mammalian mt genomes were significantly correlated with the duration of single stranded
state of heavy-strand genes during replication, with increased duration reflecting,
in part, the proximity of protein encoding genes to the origin of replication of the
heavy strand (ORH), as well as their position relative to the OR of the light strand (ORL). Longer durations in the single stranded condition increase the vulnerability of
DNAs to hydrolytic and oxidative damage creating the compositional asymmetries between
the heavy and light strands (or leading and lagging strands). In vertebrates, the
ORL is typically located two-thirds of the way around the genome from the ORH. In insects, the only protostomes examined in detail, however, the replication origins
for both strands (leading and lagging) appear to be in close proximity, near the ends
of a conserved block within the A+T rich region, with the lagging strand beginning
after 95% of the leading strand has been replicated [60]. Comparative studies are lacking for molluscs.

If the ORs for both strands are located close to one another in molluscs, then examining
nucleotide composition in relation to the position of each protein-encoding gene could
reveal putative sites for the OR. We tested this by plotting nucleotide composition
at 4FD third codon positions as in [45] versus the midpoint position of each protein-encoding gene (Figure 4). By moving the putative OR between different protein-encoding genes, we found a
compelling linear relationship, with a negative slope for %T and %G and a positive
slope for %A and %C, when we positioned the OR between nad2 and nad1 (or between nad2 and nad6 for D. gregarium). This region generally encompasses the cassette of 7 tRNA genes encoded on the "-"
strand, two rRNA genes, as well as additional tRNAs, albeit with some notable differences
amongst taxa (Figure 1). This relationship is shown based on averaged values across all four complete vermetid
genomes (Figure 4: diamonds), and separately for the genome of Dendropoma maximum (Figure 4: squares) which exhibited the strongest pattern across all four taxa. Reyes et al.
[45] found an increase in the frequency of A and C at 4FD sites in the light (sense) strand
in direct relation to the single-stranded duration of the heavy strand. They speculated
that this was the result of spontaneous deamination of adenine into hypoxanthine,
which base pairs with C rather than T, and cytosine into uracil, which base pairs
with A rather than G, along the heavy strand during its single-stranded state in replication
(with associated increases in nucleotides A and C in the light, sense strand). In
vermetid genomes, where genes are encoded on the opposite strand to vertebrates (i.e.
the heavy strand is the sense or "+" strand for all vermetid protein-encoding genes,
see Figure 3), genes with lower frequencies of G and T (or higher frequencies of C and A) at 4FD
sites should be those experiencing shorter durations in the single-stranded condition.
The marked difference in nucleotide composition of 4FD sites between nad2 (low %G and %T) and nad1 (high %G and %T) is thus suggestive of substantial differences in exposure of these
two genes to the single-stranded condition. Consequently, the OR may lie between these
two protein-encoding genes. Comparisons of nucleotide composition patterns along the
genomes of protostome taxa with well defined ORs are now necessary to confirm or refute
the predictive power of such analyses in identifying control regions.

Two other observations support the general location for the OR between nad2 and nad1. First, this region is associated with a major change in base compositional bias
reflecting the presence of rRNA genes and a cassette of tRNAs encoded on the "-" strand
(Figure 3). Such changes are thought to be associated with ORs [41,58]. Second, this region of the mt genome appears to be involved in a number of gene
order rearrangements, possibly reflecting a rearrangement "hotspot" (see Gene order rearrangements, below). Hotspots have been associated with ORs in previous studies [23]. In many other caenogastropods, however, ORs have been tentatively identified in
a different region of the genome, despite the presence of a similar cassette of tRNA
genes located on the "-" strand [48,52,53]. In these taxa, the OR is thought to be present between trnF and coxIII within an unassigned stretch of sequence of variable length (from 15 - 848 bp) associated
with inverted repeats and secondary structure elements. This gene boundary is not
present in the Vermetidae. Direct examination of mRNAs is now needed to demonstrate
conclusively the presence of ORs within vermetid and other caenogastropod mt genomes.

Gene order rearrangements

Each of the vermetid genomes examined here possessed a unique gene arrangement (Figure
5). The four complete vermetid genomes differed primarily in the position of tRNA genes,
the most mobile elements within the mt genome [16]. While the position of many tRNA genes was conserved, the location of trnA, trnK, trnP, trnT, trnLCUN, trnLUUR, and trnV was more variable across the four genomes. Only D. gregarium differed in the order of protein-encoding genes, with nad6 changing position relative to other protein encoding and rRNA genes, as described
in [18]. The two partial genomes of Vermetus erectus and Thylaeodus sp. provided additional evidence of the extent of gene rearrangement within this family,
however, with novel arrangements both tRNA and protein-encoding genes.

Figure 5.Linear arrangement of protein-encoding, rRNA and tRNA genes within the complete mt
genomes of vermetid gastropods, caenogastropods, and select representatives of other
molluscan groups. Partial genomes of Thylaeodus sp, Vermetus erectus, Littorina saxatilis and Calyptraea chinensis are also included. As is the standard convention for metazoan mt genomes, cox1 has been designated the start point for the "+" strand for all genomes. The dashed
line indicates missing genes in the representation of partial genomes. Genes are transcribed
left-to-right as depicted except for those underlined to signify opposite orientation.
Gene names are as in Figure 1 except that L1 and L2 refer to trnLCUN and trnLUUR, respectively, and S1 and S2 refer to trnSAGN and trnSUCN, respectively. Pseudogenes identified in the genomes of Dendropoma maximum and Thylacodes squamigerus are not shown. In some instances, gene orders differ from their original publications
(e.g., [53]); this is based on revised annotations listed in MitoZoa [63]. Abbreviations are as follows. GAST: Class Gastropoda; POLY: Class Polyplacophora;
CEPH: Class Cephalopoda; CA: Caenogastropoda; Li: Littorinimorpha; Ne: Neogastropoda;
VE: Vetigastropoda.

Given the extent of shared gene boundaries with representatives of other molluscan
classes, Grande et al. [15] have suggested that the mt genome of the abalone, Haliotis rubra [56], represents the ancestral gene order of gastropods, albeit with two derived changes.
The position of trnD and trnN are likely autapomorphies of Haliotis, since many caenogastropods share a different (and presumed ancestral) position for
trnD with Octopus and trnN with Octopus and Katharina (Figure 5). Based on this inferred plesiomorphic gastropod condition, vermetids exhibit a number
of derived gene order changes, some of which are shared with other caenogastropods.
All published caenogastropod mt genomes, and the four complete vermetid genomes examined
here, share an inversion of a block of 23 genes spanning from trnF to trnE in Haliotis, with a reversion (to the original strand) of genes spanning trnM to trnE within this block. As discussed by [48], this rearrangement must have occurred before the divergence of caenogastropods,
but after their separation from the Vetigastropoda (Haliotis).

Vermetid mt genomes also shared an interesting gene order change with two other members
of the superfamily Littorinimorpha, Littorina saxatilis and Oncomelania hupensis, associated with the position of the two leucine tRNA genes, trnLUUR and trnLCUN (Figure 5). Within the mt genome of Haliotis, Octopus, and Katharina, these two leucine tRNAs are sandwiched between rrnL and nad1 in the following arrangement: rrnL-trnLCUN-trnLUUR-nad1. This gene order is retained in all caenogastropod mt genomes sampled to date, except
for Littorina, Oncomelania, Dendropoma maximum, D. gregarium, and Vermetus erectus, where the relative position of these two genes has switched, such that trnLUUR is located directly upstream from trnLCUN. (Note: this gene order is incorrectly annotated in [48] (pg 40), and this gene order change is not recognized in [15](page 11) or [53](page 4), where it is stated that there are no differences in gene order between the
partial genome of Littorina and complete genome of neogastropods). This difference in the order of these two tRNAs
is also present in several other vermetid taxa (data not shown), but is not present
in two additional members of the Littorinimorpha: Cymatium parthenopeum and Calyptraea chinensis [53]. While this shared gene rearrangement may be a synapomorphy defining a clade within
the Littorinimorpha to which the Littorinidae (Littorina), Pomatiopsidae (Oncomelania) and Vermetidae belong, changes in tRNA positions involving neighbouring leucine
tRNA genes should be treated with caution. Gene translocations among neighbouring
genes appear to occur with increased frequency in mt genomes and thus may be more
likely to arise independently [23]. Such "position switches" between neighbours may therefore be less reliable phylogenetic
characters for addressing deeper level phylogenetic questions. In addition, simple
changes in position of these two leucine tRNA genes can mask more complicated dynamics
that may involve gene duplications and tRNA remolding events [28] (see Evidence for tRNA remolding and recruitment, below). Uncovering the dynamics that have occurred within this region of the genome
can be essential to interpreting gene identity correctly and accurately reconstructing
past gene rearrangement events. The discovery of two trnLUUR genes side by side between rrnL and nad1 of Thylacodes squamigerus further suggests that such tRNA remolding events may be at play within vermetid mt
genomes.

Some gene order arrangements were unique to the Vermetidae (Figure 5). All four complete vermetid genomes shared a block of three protein-encoding genes
and 3 - 4 tRNA genes: trnN-nad5-trnK-trnA-cox3-trnS1-nad2, with trnA and trnK absent from this block in Eualetes and D. gregarium, respectively. This gene rearrangement was associated with the movement of nad5 from its conserved position between trnF and trnH in Katharina, Octopus, Haliotis, and other caenogastropods, and with the break-up of cox3, trnS1, and nad2 from their conserved association within a block of genes including cox3 - 5 tRNAs - nad3 - trnS1 - nad2 in these same taxa. TrnD also shared a unique derived position between cox1 and atp8 in the Vermetidae. While trnD in Haliotis has differing neighbouring genes, the gene order cox1-trnD is shared with the chiton, Katharina, and trnD-atp8 is shared with Octopus, as well as all other caenogastropods. The derived vermetid arrangement thus appears
to be associated with a switch in the position of cox2 relative to cox1, trnD, and atp8, along a lineage leading to the Vermetidae. In addition, the vermetids also shared
a derived change in the relative positions of trnY and trnM within the cassette of 7 tRNA genes encoded on the "-" strand. Such distinctions between
the mt genome of vermetids and other littorinimorphs are particularly relevant here
given that there is both molecular and morphological support for the inclusion of
littorines and vermetids in a clade of lower caenogastropods [28,37,38]. Gene rearrangements shared by vermetids to the exclusion of other Littorinimorpha
(Littorina, Oncomelania, Calyptraea, and Cymatium), thus can be inferred to have occurred following the divergence of the ancestral
vermetid from the common ancestor of these taxa.

Numerous gene order changes have also occurred within the family Vermetidae (Figure
5). The Vermetidae has a fossil record extending to the Late Cretaceous, suggesting
that this gene reshuffling has happened within the past 65 million years, and based
on molecular dating, perhaps within the past 38 million years [18]. Preliminary evidence suggests that the mt genome of Dendropoma maximum represents the ancestral gene order of the Vermetidae. The translocation of trnK1 and trnP-nad6 found in Dendropoma gregarium has also been shown to be a derived gene order change within this genus [18]. Relative to D. maximum, most gene rearrangements evident in the three other complete genomes have occurred
between trnR and trnT. Eualetes differs from D. maximum in the position of four tRNAs: trnA, trnP, trnLUUR and trnT, with gene remolding events between trnLCUN and trnLUUR likely associated with gene order changes involving these two isoaccepting tRNA genes
(see Evidence for tRNA remolding and recruitment, below). Differences between the gene arrangement of Dendropoma maximum and Thylacodes appear related to a gene duplication event in Thylacodes spanning rrnS to nad1, as inferred from gene vestiges of rrnS, rrnL, and nad1 and extra tRNA genes (trnV, trnLUUR). Details of this rearrangement will be examined further in another paper (Rawlings
et al. in prep). Many of the conserved gene boundaries described above for the four complete
vermetid genomes sequenced, even those shared with other caenogastropods, Haliotis, Katharina, and Octopus, were not evident in the partial genomes of V. erectus and Thylaeodus (Figures 1 &5). The scale of these rearrangements is impressive given that these changes have happened
within a family of gastropods, and suggests that further sampling of the Vermetidae should
uncover more changes and perhaps intermediate stages that may reveal the dynamics
underlying the gene rearrangements shown here. The genome of Thylacodes squamigerus provides hope of this as vestiges of once functional genes discovered between annotated
genes are helping to understand the mechanism of gene order change and the appearance
of duplicated tRNAs. This expectation has also been borne out by additional data collected
as part of a phylogenetic analysis of the Vermetidae: numerous gene order changes
have now been uncovered within vermetid taxa based on short sequences (<3.5 kb) extending
from rrnS to nad1 (Rawlings et al., in prep; data not shown). With further sampling of the Vermetidae, we anticipate
that these gene order rearrangements will provide a suite of robust characters that
can be used, in addition to morphological and nucleotide sequence characters, to build
a well-supported phylogenetic hypothesis for this family and to firmly place the Vermetidae
within the context of caenogastropod evolution.

Evidence for tRNA remolding and recruitment

Implicit in the use of secondary structure characteristics and anticodon triplets
to recognize tRNAs is the assumption that tRNA genes cannot change identity by simple
nucleotide substitutions in their anticodon. For the most part this seems to be true
within animal mt genomes, likely because of the presence of specific recognition elements
that are required by tRNA synthetases to identify and correctly charge their associated
tRNAs. However, evidence is accumulating that tRNAs do occasionally change identities.
Through a process known as tRNA remolding, identity change does occur between the
two isoaccepting leucine tRNA genes [25,26,28] and possibly also between the two isoaccepting serine tRNA genes [61]. Cases of tRNA identity change across non-isoaccepting tRNAs, referred to as tRNA
recruitment, are now also coming to light [27]. The strongest evidence of tRNA remolding is the discovery of unexpectedly high levels
of sequence similarity between two tRNA leucine genes [28]. Consequently, these dynamics are most easily recognized when they have happened
recently, before mutational changes can obscure the common history of the duplicated
tRNAs. Although gene remolding and recruitment events are often associated with changes
in gene order, the pattern of duplicate loss may result in maintenance of the original
gene order. In fact, recognizing gene remolding events can often help to uncover genome
dynamics that are hidden at the level of gene order alone [28].

Given that leucine tRNAs seem particularly susceptible to remolding events [26,28], we investigated the sequence similarity between both leucine tRNAs within each vermetid
taxon as well as other select caenogastropods, Haliotis, Octopus, and Katharina (Table 9). High sequence similarities (80% or greater) between leucine tRNA genes were found
in five taxa (Table 9) suggesting that one tRNA has taken over the role of the other through a process
of gene duplication, mutation in the anticodon triplet and the eventual loss of the
original gene [26,28]. The T. squamigerus genome represents a particularly interesting case of this. Within this genome, the
two copies of LUUR only share 67.6% sequence identity; in comparison there is 98.4% sequence similarity
between trnLCUN and trnLUUR2. Consequently, we can assume that trnLUUR2 is a duplicated copy of trnLCUN that has subsequently undergone a mutation in the third position of the anticodon
to assume the identity of trnLUUR. Such events argue for the exclusion of tRNA leucine genes in gene-order based phylogenetic
analyses at high taxonomic levels; at low taxonomic levels, such as within the Vermetidae,
however, they offer the promise of new phylogenetic characters and the discovery of
gene dynamics that may not be evident at the level of gene order alone. Given the
importance of identifying remolding events, it is surprising that so little attention
is often paid to the dynamics of these two leucine tRNA genes. Failure to distinguish
between these two genes correctly can also lead to mistakes in interpretation, where
two genomes are considered to have identical gene orders, but differ in the position
of these two leucine tRNAs (see comparisons between Littorina saxatilis and other caenogastropods in [15,48,53]).

Table 9. Percentage similarity in nucleotide sequences between isoaccepting leucine tRNA genes
(trnLUUR and trnLCUN) within the mt genomes of six vermetids, sixteen additional caenogastropods, and
three other molluscs for comparison

The presence of a second trnK within the genome of Dendropoma gregarium is suggestive of a past duplication event within this genome, likely the result of
slipped-strand mispairing during replication. The sequence similarity between the
two lysine tRNAs (trnK) is not high (38.0%). Comparisons between these sequences and the sequences of presumed
trnK orthologs from other taxa revealed strong similarities between these and the trnK1 located between trnV and trnP (Figure 6). The second trnK (trnK2), located between trnI and nad3 lacked many of the conserved sequence elements present in other trnKs (Figure 6). We compared trnK2 with other tRNAs within the genome of D. gregarium to determine if this might represent a case of tRNA recruitment [27], but did not uncover any strong matches with any other gene. Consequently, the origin
and function of this putative tRNA are currently unclear.

Figure 6.Structural alignments of lysine tRNA genes sampled from four vermetid gastropods and
other select caenogastropods. Two tRNA genes are included from Dendropoma gregarium, one located between trnV and trnP (trnK1), the other located between trnI and nad3 (trnK2). Nucleotides appearing in >50% of taxa at a given position in the gene are shaded
grey. Colors below each region of the alignment are used to show the associated position
in the colored tRNA secondary structure model.

Conclusions

Here we have presented gene order and sequence characteristics for six gastropods
within the family Vermetidae, including the complete mt genomes of four species (Dendropoma maximum, D. gregarium, Eualetes tulipa, and Thylacodes squamigerus) and the partial mt genomes of two others (Vermetus erectus and Thylaeodus sp.). The publication of these genomes increases the number of complete caenogastropod
mt genomes sequenced from 16 to 20 and presents the first direct evidence of major
gene order rearrangements within the Littorinimorpha.

While molluscs have long been recognized as having unusually dynamic mt genomes, preliminary
sampling of the Caenogastropoda represented by genomes from 16 species has suggested
a model of highly conserved mt gene order. Our results reverse this trend and provide
further evidence of the lability of the molluscan mt genome by describing extensive
gene order changes that have occurred within one family of caenogastropod molluscs,
the Vermetidae. Based on these results, we anticipate that the mt genomes of caenogastropods
exhibit a variety of gene order arrangements, which, if explored and exploited, could
provide a wealth of phylogenetically-informative characters to enhance our understanding
of the evolutionary radiation of this diverse clade of gastropods.

Despite the extent of mt gene rearrangement within the Vermetidae, their genomes exhibit
similar characteristics to other caenogastropods. Each complete genome contained the
full complement of 37 genes, although additional tRNA genes were evident in the mt
genomes of D. gregarium (trnK) and Thylacodes squamigerus (trnV, trnLUUR). Protein-encoding and rRNA genes were all encoded on the same strand (i.e. the "+"
strand), not unusual for caenogastropods, whereas tRNA genes, the most mobile mt genomic
components, were distributed between the "+" and "-" strands. Nucleotide skews patterns
(i.e. AT and GC-skews) of vermetid genomes and their various components were similar
to those described for other caenogastropods, although the AT bias was less pronounced
than other caenogastropods described to date. Although no control regions were definitively
identified, a compelling trend in the relationship between nucleotide composition
and position from nad1 was uncovered, which appears worthy of further investigation.

Our results also demonstrate that focused sampling of mt genomes at low taxonomic
levels can be extremely productive, both in terms of uncovering characters useful
in phylogeny and in understanding more fully the evolutionary dynamics and mechanics
of mt gene rearrangements. Each of the six vermetid mt genomes examined had a unique
gene order, with evidence of gene rearrangements involving translocations of tRNA
and protein-encoding genes, and one gene inversion, occurring within this family.
Additional rearrangements have also been uncovered amongst other representatives of
this family suggesting that further sampling of complete mt genomes within the Vermetidae
will also prove useful. The extent of gene rearrangement within such an evolutionarily
young group offers the opportunity to explore gene dynamics such as tRNA remolding,
which appears to have been rife within this family, and to investigate its consequences
for phylogenetic analyses based on gene orders. The sampling of mt genomes, such as
that of Thylacodes squamigerus, in the intermediate stages of a gene rearrangement, may also help to illuminate
mechanisms of gene translocation and inversion and to interpret past events that have
shaped an organism's current mt gene order. Continued studies of the mt genome dynamics
within the Vermetidae will improve our understanding of this family's phylogeny, its
phylogenetic placement within the Littorinimorpha, and the mechanisms and processes
that have acted to shape the mt genomes of this family and other metazoans.

Authors' contributions

RB and TAR obtained the samples. TAR and TMC extracted the DNA and amplified the genomes
which were then sequenced by JLB. TAR and MJM annotated the genome, MJM undertook
the descriptive analyses and TAR and MJM wrote the first draft of the manuscript.
All authors participated in subsequent revisions of the manuscript.

Acknowledgements

We thank Drs. Michael Hadfield, Isabella Kappner, Jon Norenburg, and Richard and Megumi
Strathmann for vermetid samples, and Dr. Mónica Medina for assistance with genome
sequencing. MJM was funded in part by CBU RP Grant 8341 to TAR. Vermetid research
was supported by NSF DEB(REVSYS)-0841760/0841777 to RB, TMC, and TAR.

Proceedings of the Royal Society of London Series B-Biological Sciences 2004, 271(1538):537-544. Publisher Full Text

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